Project 3 - Mapping Long-range Allosteric Pathways in CRISPR-Cas9

项目 3 - 绘制 CRISPR-Cas9 中的长程变构途径

• 批准号: 10271625
• 负责人: GEORGE LISI
• 金额: $ 37.74万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10271625
• 关键词: Active Sites; Allosteric Regulation; Amino Acids; Binding; Biochemical; Biological; Biological Assay; Biological Models; Biological Process; Biomedical Engineering; Biophysics; CRISPR/Cas technology; Cell physiology; Centers of Research Excellence; Chemicals; Clustered Regularly Interspaced Short Palindromic Repeats; Communication; Community Networks; Computational Biology; Computer Simulation; Computing Methodologies; Coupling; Crystallization; DNA; DNA Binding; Data; Disease; Engineering; Ensure; Enzymes; Equilibrium; Gene Targeting; Genome; Genomics; Guide RNA; Investigation; Knowledge; Laboratories; Length; Link; Maps; Methods; Modernization; Modification; Molecular; Molecular Biology; Molecular Conformation; Motion; Mutation; Nuclear Magnetic Resonance; Nucleotides; Pathogenicity; Pathology; Pathway Analysis; Pathway interactions; Play; Process; Protein Biochemistry; Proteins; RNA Binding; Regulation; Regulator Genes; Relaxation; Roentgen Rays; Role; Signal Pathway; Signal Transduction; Site; Specificity; Structure; Tertiary Protein Structure; Therapeutic; Work; base; biophysical analysis; computer studies; design; drug discovery; ds-DNA; enzyme mechanism; experimental study; flexibility; genome editing; human disease; improved; in vivo; insight; interdisciplinary approach; millisecond; molecular dynamics; mutant; novel; novel strategies; nuclease; precision medicine; protein function; recruit; response; scaffold; simulation; small molecule inhibitor; success; theories; therapeutic enzyme; tool

Project Summary Gene regulatory mechanisms are critical for proper cellular and protein function, and modern molecular biology has linked numerous pathologies to dysregulation of these processes. Although modification of the genome to correct pathogenic mutations is a promising therapeutic approach, these efforts cannot be successful without knowledge of the underlying biochemistry of protein machinery such as CRISPR- Cas9 (Cas9). Cas9 can be a customizable tool to edit and correct disease-linked (genomic) mutations, however, to fully realize these applications, novel strategies to overcome its off-target effects and poor temporal control must be investigated. Cas9 utilizes a guide RNA molecule to recruit, stabilize, and facilitate cleavage of double-stranded DNA after recognition of a well-known protospacer adjacent motif (PAM) sequence. Prior X-ray crystal structures indicate that conformational changes within the Cas9 nucleases, HNH and RuvC, are required for effective catalytic function. However, these structures offer little mechanistic information, as the target DNA and catalytic nucleases are never observed in an activated state. The conformational shift of HNH, in particular, is correlated to motions of neighboring subdomains, all of which are activated from >20 Å away by the PAM-binding domain, suggesting an allosteric mechanism. Understanding this allosteric coupling would have exciting potential for precision medicine by establishing novel paradigms to control and enhance the spatial and temporal function of Cas9. We recently identified a pathway of millisecond timescale motions spanning the HNH nuclease and reaching multiple Cas9 domains that computational results suggest is a portion of a larger allosteric network that controls Cas9 function. To investigate the reach of this allosteric network and the role of molecular motions in its mechanism, my laboratory will undertake a synergistic solution NMR and computational study to map the long-range allosteric pathway of Cas9. We will now (1) characterize allosteric mutants of HNH that are known to alter Cas9 specificty, (2) establish the biophysical roles of the neighboring REC2 and REC3 domains in propagating allosteric signals to/from HNH, and (3) characterize the conformational ensemble governing the full-length Cas9 protein. This multidisciplinary approach of NMR spin relaxation experiments, molecular dynamics simulations, and network theory, will probe multi-timescale protein motions in Cas9, revealing specific amino acids responsible for transmitting structural or dynamic information. These studies will use both full-length Cas9 and novel engineered constructs to interrogate specific domains within the 160 kDa enzyme. The structural and dynamic findings of this work will be correlated to function with new in vivo assays to provide a detailed understanding of the Cas9 allosteric mechanism.

项目摘要 基因调节机制对于适当的细胞和蛋白质功能至关重要，现代分子 生物学将许多病理与这些过程的失调联系起来。虽然修改 纠正致病突变的基因组是一种有希望的疗法，这些努力不能 在不了解蛋白质机械的基本生物化学（例如CRISPR-）的情况下取得成功 CAS9（CAS9）。 CAS9可以是编辑和纠正疾病连接（基因组）突变的可自定义工具， 但是，为了充分实现这些应用，要克服其脱靶效应和差的新型策略 必须研究临时控制。 CAS9利用指南RNA分子募集，稳定和 在识别众所周知的蛋黄邻近基序后，促进双链DNA的裂解 （PAM）序列。先前的X射线晶体结构表明CAS9内的会议变化 有效的催化功能需要核酸酶HNH和RUVC。但是，这些结构提供 几乎没有机械信息，因为在一个目标中从未观察到目标DNA和催化核。 激活状态。特别是HNH的会议转变与邻近的动作有关 亚域，所有这些都从> 20Å远的pam结合域激活，这表明 变构机制。了解这种变构耦合将具有精确的激动人心的潜力 通过建立新的范式来控制和增强新型范式来控制和增强的空间和临时功能 Cas9。我们最近确定了跨越HNH核酸酶的毫秒时尺度运动的途径 并达到多个CAS9域，计算结果表明是较大变构的一部分 控制CAS9功能的网络。调查这种变构网络的影响及 我的实验室将在其机理中进行分子运动，将进行协同解决方案NMR，并进行 绘制CAS9的远程变构途径的计算研究。我们现在（1）描述 已知会改变CAS9特异性的HNH的变构突变体，（2）建立生物物理作用 在向HNH传播变构信号时，相邻的REC2和REC3域，（3） 表征统治全长cas9蛋白的构象合奏。这个多学科 NMR自旋松弛实验，分子动力学模拟和网络理论的方法将 探针在Cas9中的多次量度蛋白质运动，揭示了负责传输的特定氨基酸 结构或动态信息。这些研究将使用全长Cas9和新颖的工程 构造以询问160 kDa酶内的特定域。结构和动态 这项工作的发现将与新的体内测定法相关联，以提供详细的 了解CAS9变构机制。